Novel antibody recognizing a small subset of human hematopoietic cells

ABSTRACT

The subject invention pertains to antibodies that have binding specificity for an antigen that is expressed on a subset of human, hematopoietic mononuclear cells, including a hematopoietic stem cell population, but is not expressed on normal, mature myeloid cells. In one embodiment, a monoclonal antibody, MG1, is provided. This antibody is useful in methods of isolating cell suspensions from human blood and marrow that can be employed in bone marrow transplantation, genetic therapy, and in treating other diseases of the hematopoietic system. Cell suspensions containing MG1 +  human hematopoietic cells are also provided, as well as therapeutic methods employing the cell suspensions. The subject invention also pertains to the novel antigen recognized by the subject antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No.09/531,827, filed Mar. 21, 2000, now U.S. Pat. No. 6,242,579, which is adivision of U.S. application Ser. No. 08/970,032, filed Nov. 13, 1997,now U.S. Pat. No. 6,043,348, which claims the benefit of provisionalapplication U.S. Ser. No. 60/030,428, filed Nov. 13, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel monoclonal antibodyrecognizing a small subset of human hematopoietic cells, which mayinclude the hematopoietic stem cell population.

BACKGROUND OF THE INVENTION

[0003] Hematopoietic stem cells are defined as those cells that arecapable of both self-renewal and differentiation into the two principleprecursor components—the myeloid and lymphoid lines. Such stem cells aresaid to be “totipotent.” Stem cells that are less general but that canstill differentiate into several lines are called “pluripotent.” Furtherdifferentiation then occurs among the precursor cells to produce themonocyte, eosinophil, neutrophil, basophil, megakaryocytes, anderythroid lineages from the myeloid line, and T cells, B cells, and NKcells from the lymphoid line. For a background review of the stem cellsee Scientific American 256:86-93 (December 1991).

[0004] One of the first breakthroughs into stem cell isolation andidentification came in the late 1980's. In U.S. Pat. No. 4,714,680 (Dec.22, 1987), Civin described a population of pluripotentlympho-hematopoietic cells that were substantially free of maturelymphoid and myeloid cells. Civin also described an antigen, MY-10, anda monoclonal antibody thereto, which was present on those cells. Thosecells made up about 1% of all cells in normal adult bone marrow, andgenerally comprised a mixture of totipotent, and pluripotent stem cellsand lineage committed precursor cells with the latter cellspredominating.

[0005] Since that time, MY-10 has been classified by the InternationalWorkshop on Human Leukocyte Antigens as falling with the clusterdesignated as “CD34.” Anti-CD34 monoclonal antibodies are nowcommercially available from a number of sources including, for example,Becton Dickinson Immunocytometry Systems (“BDIS”).

[0006] Anti-CD34 monoclonal antibodies have been used for a number ofpurposes. Loken, Terstappen and their collaborators have published aseries of papers describing the maturational stages for variouscomponents of the hematopoietic system, such as B lymphocytes (Loken etal., Blood 70:1316-1324 (November 1987)), erythroid cells (Loken et al.,Blood 69:255-263 (January 1987)), and neutrophils (Terstappen et al.,Leukemia 4:657-663 (September 1990)). The objective of these studies wasto define, starting from the most mature cell and working backwards, thevarious maturational and developmental stages of a lineage committedcell.

[0007] Anti-CD34 monoclonal antibodies have also been used to look forearlier non-lineage committed stem cells. For example, Terstappen etal., Blood 77:1218-1227 (March 1991), described a subset of humanprogenitor cells that were capable of self-renewal and differentiationinto each of the various hematopoietic lineages (i.e., a population ofcells that include cells that are totipotent). This population wascharacterized as being CD34⁺/CD38⁻.

[0008] U.S. Pat. No. 5,061,620 to Tsukamoto et al (Oct. 29, 1991) alsodescribed a population of cells that were capable of self-renewal anddifferentiation. This population of cells was characterized as beingCD34⁺/CD10⁻/CD19⁻/CD33⁻ and Thy-1⁺.

[0009] Other investigators have attempted to subset CD34⁺ cells fromboth peripheral blood and bone marrow. Bender et al., Blood 77:2591-2596(June 1991), used four color flow cytometry with combinations ofmonoclonal antibodies (i.e., anti-CD34, anti-CD33, anti-CD45, anti-CD19,anti-CD7, anti-CD10, anti-CD3, anti-CD20, anti-CD 14, anti-CD11b andanti-HLA-DR), to identify and isolate CD34⁺ hematopoietic progenitorcells. Bender et al. were able to identify a number of subsets. Onesubset was CD34⁺/HLA-DR−. This subset had a very small number of cellsand no clear population of this phenotype was resolved. Bender et alspeculated on the ability of this population of cells to give rise toblast cell colonies or cells reconstituting long term cultures basedupon prior work of others.

[0010] Sutherland et al., Blood 78:666-672 (August 1991), reported onthe differential regulation of “primitive” hematopoietic cells in longterm culture. They used anti-CD34 and anti-HLA-DR monoclonal antibodiesto select cells that were CD34⁺ and HLA-DR^(dim) or HLA-DR⁻. These cellswere then grown on a unique stromal cell line. The purpose of this workwas to establish a method of long term culture of such cells for thepurposes of studying hematopoiesis and the effect of different growthfactors on hematopoiesis.

[0011] Simmons et al., Blood 78:55-62 (July 1991), also reported on the“identification” of a stromal cell precursor in human bone marrow. Usingan antibody they designated “Stro-1,” Simmons et al. were able to removestromal cells from bone marrow. The antigen recognized by this antibodywas not present on colony forming progenitor cells but was present on a“subpopulation of cells experiencing the [CD34] antigen.” Thus, Simmonset al. described the ability of the antibody to separate out stromalcells from hematopoietic cells in bone marrow before culture.

[0012] Verfaillie et al, J. Exp. Med. 172:509-520 (August 1990),reported on a CD34⁺/HLA-DR⁺ and CD34⁺/HLA-DR⁻ population of “primitive”progenitor cells. Taking adult marrow, Verfaillie et al. depleted bonemarrow of lineage⁺ cells using multiple monoclonal antibodies. Next,fluorescently labeled CD34 and HLA-DR monoclonal antibodies were used toselect HLA-DR⁺ and HLA-DR⁻ populations that were also CD34⁺. Havingisolated these two groups, Verfaillie et al. reported that the HLA-DR⁺cells were better in short term culture than the HLA-DR⁻ cells. In longterm culture, the reverse was true.

[0013] WO 93/25216, published Dec. 23, 1993, teaches a population ofhuman primitive stem cells that are capable of self-renewal and that arecapable of differentiating into hematopoietic stem cells and stromalstem cells that give rise to the hematopoietic microenvironment. Thispopulation of cells has the phenotype CD34⁺/CD38/HLA-DR. This populationof cells lacks lineage committed antigens (i.e., is CD33−, CD10−, CD5−,and CD71−). Cells having this phenotype were identified in adult andfetal peripheral blood, bone marrow, thymus, liver, or spleen using acombination of antibodies and selecting for the presence or absence ofthe antigens recognized by these antibodies on the cells. Preferably,the combination of antibodies comprised at least three monoclonalantibodies and more preferably comprised anti-CD34, anti-CD38 andanti-HLA-DR monoclonal antibodies.

[0014] WO 94/02157, published Feb. 3, 1994, teaches the isolation ofhuman hematopoietic stem cells that are CD34⁺, HLA-DR and express thereceptor for the c-kit ligand (KR⁺). This cell population was reportedlyuseful for transplantation and in gene therapy protocols.

[0015] To date, the CD34 antigen, as identified by monoclonalantibodies, has been the only known cell surface marker to be used todefine the hematopoietic stem cell compartment and has become the markerof choice not only for the identification of stem cells but also fortheir isolation. Published information now indicates the existence ofmonoclonal antibodies that define cell surface markers distinct fromCD34; (i) monoclonal antibody AC 133 which binds to a surface protein of96 kDa on approximately 50% of CD34⁺ cells; (ii) monoclonal antibody BB9which binds to a surface protein of 160 kDa on approximately 10-28% ofCD34⁺ cells; and (iii) a non-designated monoclonal antibody that bindsto a glycoprotein 105 on the surface of hematopoietic stem cells.Virtually all of the CFU-S and colony forming unit cells detectable byin vitro stem cell assays express the CD34 antigen. Furthermore, anumber of animal and human studies have demonstrated that purified CD34⁺cells are capable of reconstituting the entire hematopoietic system,suggesting that early engraftment by progenitor cells and long-termmaintenance by primitive stem cells are mediated by this population(See, e.g., Berenson et al., J. Clin. Invest. 81:951-955 (1988)).

[0016] The identification and isolation of the most primitive populationof hematopoietic stem cells would be highly advantageous in situationswhere reinfusion of only a small number of long-term repopulating cellswas desired. For example, this would be the case when purging bonemarrow or peripheral blood stem cells of contaminating tumor cells, orwhere genetic manipulation of the stem cells was the objective. CD34expression seems to be stage specific rather than lineage specific withhigher levels of expression seen in primitive progenitors and decreasingexpression levels with cellular maturation (Holyoake & Alcom, Blood Rev.8(2):113-124 (1994)). Nonetheless, it has never been successfullydemonstrated that stem cells could be purified on the basis of theirCD34 expression levels. The studies described above suggest that CD34⁺cells selected for the absence of lineage specific markers, such as,CD33, CD38, HLA-DR, as well as low Thy-1 labeling (Thy^(lo)) (Craig etal., J. Exp. Med. 177:1331-1342 (1993)), correspond to a stemcell-enriched population. No positive enrichment procedure, however, hasever been described.

[0017] Clearly, there is a continuing need in the art to isolate novelmarkers of primitive stem cell populations so that positively enrichedprimitive stem cell populations can be obtained and utilized astherapeutic compositions, as well as in therapeutic methods such as bonemarrow transplantation and gene therapy.

[0018] Bone marrow transplantation is an effective therapy for anincreasing number of diseases. Graft Versus Host Disease (GVHD),however, limits bone marrow transplantation to recipients withHLA-matched sibling donors. Even then, approximately half of theallogenic bone marrow transplantation recipients develop GVHD. Currenttherapy for GVHD is imperfect and the disease can be disfiguring and/orlethal. Thus, risk of GVHD restricts the use of bone marrowtransplantation to patients with otherwise fatal diseases, such asmalignancies, severe aplastic anemia, and congenital immunodeficiencystates.

[0019] The potential benefits from expanded use of bone marrowtransplantation have stimulated research on the cause and prevention ofGVHD. It has been shown that donor T lymphocytes cause GVHD in animals.Removal of T lymphocytes from donor marrow inocula (“grafts”) preventedthe subsequent development of GVHD in mice, dogs, and monkeys. Similartrials in humans with monoclonal antibodies against human T lymphocytesare now in progress. Preliminary results, however, suggest onlyattenuation of GVHD, not a cure. Similar results have been achieved withE-rosette and soybean lectin depletion of T lymphocytes. Anotherapproach under investigation is the use of anti-T lymphocyte monoclonalantibodies conjugated to toxins, such as ricin.

[0020] As of yet, however, GVHD has not been prevented or cured in bonemarrow recipients. Therefore, a continuing need exists for improvedmethods of combating Graft Versus Host Disease.

[0021] Donors of bone marrow are also faced with undesirable proceduresand risks. The current procedures for harvesting bone marrow areexpensive and painful. Furthermore, the current donation procedure isaccompanied by the risks associated with anesthesia, analgesia, bloodtransfusion and possible infection. It would be desirable, therefore, toimprove the current method of harvesting hematopoietic stem cells fromdonors.

BRIEF SUMMARY OF THE INVENTION

[0022] The present invention concerns an antibody that recognizes asmall subset of human hematopoietic mononuclear cells, which may includethe hematopoietic stem cell population. An exemplified embodiment of anantibody of the invention is the MG1 monoclonal antibody.

[0023] The MG1 antibody recognizes an antigen on a small subset of humanhematopoietic mononuclear cells, but does not bind to antigens onnormal, human mature myeloid cells. The invention also concerns thehybridoma which produce the MG1 antibody.

[0024] The present invention also concerns a method for preparing a cellpopulation useful for stem cell transplantation that is positivelyenriched in immature marrow cells and substantially free of maturemyeloid and lymphoid cells.

[0025] The present invention also pertains to a method of collectingdonations useful for stem cell transplantation that avoids thedisadvantages of conventional marrow harvesting techniques.

[0026] The present invention also concerns a therapeutic materials andmethods for transplanting stem cells that can extend the use of stemcell transplantation to the treatment of non-fatal diseases.

[0027] The present invention also provides a method of stem cell genetherapy, utilizing antibodies of the present invention.

[0028] The present invention also pertains to materials and methods toreduce or eliminate GVHD associated with bone marrow transplantation.

[0029] In one embodiment, the present invention provides a method ofselecting a population of human cells containing MG1⁺ hematopoieticcells comprising: (a) providing a cell suspension from human tissue,such as marrow or blood; (b) contacting said cell suspension with anantibody that binds the MG1 antigen; and (c) separating and recoveringfrom said cell suspension the cells bound by said antibody.

[0030] In a further embodiment, the present invention provides a methodof selecting a population of human cells containing MG1⁺ hematopoieticcells comprising: (a) providing a cell suspension from human tissue,said tissue selected from the group consisting of marrow and blood; (b)contacting said cell suspension with a solid-phase linked MG1 monoclonalantibody; (c) separating unbound cells from solid-phase linkedmonoclonal antibody after said contacting; and (d) recovering boundcells from said solid-phase linked monoclonal antibody after separatingsaid unbound cells.

[0031] Yet another embodiment of the present invention provides asuspension of human cells comprising MG1⁺ hematopoietic cellssubstantially free of mature cells, as well as therapeutic methodsemploying such a cell suspension.

[0032] In a further embodiment, the present invention provides a methodof gene therapy utilizing the monoclonal antibody of the presentinvention to select for hematopoietic cells that express the MG1antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The designation “Miki,” appearing in any of the Figures submittedherein, and described below, is simply the former designation for “MG1,”and thus, should be considered the same as MG1.

[0034]FIG. 1 is a flow chart describing the origin of the MG1 hybridomaclone.

[0035]FIG. 2 shows an SDS PAGE of purified MG1 IgG. Different volumes ofde-salted, purified MG1 immunoglobulin were analyzed by 10% SDS-PAGE.The gel was fixed, and stained with coomassie blue, and photographed. 2μl, 5 μl, 10 μl and 15 μl indicate the volumes of purified MG1immunoglobulin loaded per well. Marker: BioRad low range prestainedmarkers: the positions of the 106, 80, 49, and 32 kDa bands areindicated.

[0036]FIG. 3 shows MG1 antigen expression on the surface of variouscells lines. ML-1 (NSC1.1), KG1a, K562, HT 29, and Hel cells wereincubated with MG1 hybridoma supernatant, then fluorescently labeledwith goat anti-mouse FITC-labeled polyclonal antibody, and examined forthe presence of surface labeling by flow cytometry.

[0037]FIG. 4 shows a flow cytometry analysis of whole human bone marrowusing fluorescein-labeled MG1 monoclonal antibody. Whole normal adulthuman bone marrow was labeled with MG1-FITC and anti-CD34-PE (QBend 10),fixed, and analyzed by flow cytometry. 50,000 events were collected. Thex-axis depicts MG1-associated fluorescence intensity. The y-axis depictsCD34-associated fluorescence intensity. MG1 positive cells (lower rightquadrant) represent less than 1% of all bone marrow cells, whereas,CD34+ cells (upper left quadrant) represent approximately 1% of all bonemarrow cells. FIG. 4 shows that MG1 recognizes only a very smallpopulation (less than 1%) of cells within bone marrow, and that lessthan 3% of CD34+ cells co-express MG1 antigen, suggesting a very smalloverlap of the two populations. Quad, quadrant; UL, upper left; LR,lower right.

[0038]FIG. 5 shows hybridization of Western blot with MG1 antibodyshowing the approximate molecular weight of the MG1 antigen. Cellextracts from 106 cells, prepared from various cell lines, wereseparated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membrane was probed with MG1 primaryantibody (hybridoma supernatant at a 1:100 dilution), and HRP-conjugatedsheep anti-mouse IgG secondary antibody, and visualized by ECL. Marker:BioRad high range prestained markers; the positions of the 205, 116, and80 kDa bands are indicated with short lines. ML-1; NSC1.1 cell extracts.HT-29, HEL, K562, and KG1a, refer to cell extracts from the respectivecell lines.

[0039]FIG. 6 shows hybridization of Western blot with MG1 hybridomasupernatant. Cell extracts from 10⁶ cells, prepared from various celllines, were separated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membrane was hybridized to MG1 primaryantibody (hybridoma supernatant at a 1:10 dilution), and HRP-conjugatedsheep anti-mouse IgG secondary antibody, and visualized by ECL. Marker:BioRad low range prestained markers: the positions of the 106, and 80kDa bands are indicated with hyphens. ML-1; NSC1.1 cell extracts. HT-29,HEL, K562, and KG1a, refer to cell extracts from the respective celllines.

[0040]FIG. 7 shows hybridization of Western blot with MG1 purifiedantibody. Comparison with FIG. 6 shows that the purified antibody andhybridoma supernatant recognize the same protein in cell extracts. Cellextracts from 10⁶ cells, prepared from various cell lines, wereseparated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membrane was hybridized to MG1 primaryantibody (purified IgG), and HRP-conjugated sheep anti-mouse IgGsecondary antibody, and visualized by ECL. Marker: BioRad low rangeprestained markers: the positions of the 106, and 80 kDa bands areindicated with hyphens. ML-1; NSC1.1 cell extracts. HT-29, HEL, K562,and KG1a, refer to cell extracts from the respective cell lines.

[0041]FIG. 8 shows hybridization of blood cell extracts with MG1antibody. Cell extracts from NSC1.1 cells or from different populationsof blood cells separated over Percoll gradients, were separated by SDSPAGE through 10% gels, and transferred to nitrocellulose membranes. Themembrane was probed with purified MG1 IgG as primary antibody, andHRP-conjugated sheep anti-mouse IgG secondary antibody, and visualizedby ECL. ML-1 lysate; lysate from 106 NSC1.1 cells. RBCs, Granulocytes,Lymphocytes, Thrombocytes; lysates prepared from 105 red blood cells,granulocytes, lymphocytes and thrombocytes, respectively. Marker: BioRadhigh range prestained markers; the positions of the 205, 116, and 80 kDabands are indicated as short lines.

[0042]FIG. 9 shows a Western blot demonstrating that the MG1 antigen isglycosylated. 100 ml crude NSC1.1 lysate was incubated with theindicated glycosidases at 37° C. overnight. The samples were thendiluted and separated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membranes were hybridized to MG1 primaryantibody, and HRP-conjugated sheep anti-mouse IgG secondary antibody,and visualized by ECL. Marker: BioRad high range prestained markers; thepositions of the 205 and 116 kDa bands are indicated. Control; lysatesincubated without enzyme. O-glycosidase; lysates incubated with 2.5 mUof O-glycosidase. N-glycosidase; lysates incubated with 6 unitsN-glycosidase. O+N-glycosidase; lysates incubated with both O- andN-glycosidases.

[0043]FIG. 10 shows partial purification of the MG1 antigen by affinitycolumn chromatography. Lysates prepared from NSC1.1 cells were loadedonto an immunoaffinity column containing bound purified MG1 monoclonalantibody. The column was washed, and the bound protein was eluted with0.1 M glycine (pH 2.7) into 1.0 M Tris (pH 9.0). This eluate wasconcentrated by ultrafiltration, and examined by SDS PAGE. The size ofthe MG1 antigen, as calculated by a regression analysis of the twoprotein marker lanes (non-prestained), is 186 kDa±5%. Legend: pm,prestained protein molecular weight markers; m, Novex ‘Mark 12’ proteinmolecular weight markers (The positions of the 200, 116.3, 97.4, 66.3,55.4, 36.5, and 31.0 kDa bands are indicated); lys 1, aliquot of the NSC1.1 lysate, and lys 2, aliquot of a second protein extraction of the NSC1.1 lysate; f-t, the flow-through from loading the affinity column; elu,eluate; conc. elu, concentrated eluate; re-elu, second elution from theaffinity column.

[0044]FIG. 11 shows a Western blot analysis for detection of lamininreactivity using NSC1.1 lysate and a polyclonal antibody against humanlaminin. NSC1.1 lysates (10 μl per lane), NSC1.1 conditioned medium (10μl of condition medium concentrated 10× on a Centriprep column) andpurified mouse laminin (approximately 21 μg per lane) were separated onSDS-PAGE through 10% gels and transferred to nitrocellulose membranes.The membrane was separated into three parts, and the individual partshybridized to anti-laminin polyclonal antibody (1:2000 dilution), or MG1monoclonal antibody (1/10 dilution), or PBS alone. Following incubationwith the primary antibody, the membranes were washed, and horseradishperoxidase labeled goat anti-mouse (or donkey anti-rabbit) IgG secondaryantibody was added (1/2000 dilution). Detection of antigen/antibodycomplexes was performed using the chemiluminescent reagent fromAmersham's ECL system. The resulting X-ray films were developed using aKodak M35A X-OMAT processor and the autoradiographs examined forpositive reactions. The positions of the 205, 116, and 80 kDa bands ofthe BioRad high range prestained marker are indicated with short lines.Legend: ML-1 lysate, NSC1.1 lysates; ML-1 C.M, NSC1.1 conditionedmedium; Miki, 1,2,3: lanes labeled with MG1 monoclonal primary antibody;PBS, 1,2,3: no primary antibody; polyclonal laminin Ab, 1,2,3: laneslabeled with anti-laminin polyclonal antibody.

BRIEF DESCRIPTION OF THE SEQUENCES

[0045] SEQ ID NO. 1 shows a partial amino acid sequence of the MGantigen polypeptide according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The subject invention concerns a novel polypeptide antigen. Theantigen, which is referred to herein as the “MG1 antigen,” oralternatively, the “MG1 ligand,” is expressed on a very small populationof human hematopoietic cells, which may include pluripotenthematopoietic stem cells. Stem cells have the ability to restore, whentransplanted, the production of myeloid and lymphoid cells to a patientwho has lost such production due to, for example, radiation therapy ordisease. The antigen disclosed herein is not expressed by mature myeloidcells. The newly discovered antigen may help to define a population ofhematopoietic cells desirable for use in a bone marrow transplant. Thisantigen was identified by a monoclonal antibody raised against the humanNSC1.1 cell line, which is derived from the ML-1 cell line.

[0047] The present invention also concerns antibody that can bind to theMG1 antigen. An exemplified embodiment of the invention is a monoclonalantibody, referred to herein as “MG1,” that facilitates the isolation ofthe desired cells and makes possible improved therapeutic techniquesthat significantly contribute to the understanding and prevention ofGraft Versus Host Disease. The isolated stem cells can also be employedto produce panels of monoclonal antibodies to stem cells. The monoclonalantibodies of the invention can also be employed in stem cell genetherapy.

[0048] The ML-1 cell line used as an immunogen as described herein wasderived from an enriched, immunoselected, CD34⁺ population isolated fromnormal human cadaveric bone marrow (U.S. Pat. No. 5,650,299, issued Jul.22, 1997). The immunoselected CD34⁺ cells were cultured, in vitro, inthe presence of either recombinant IL-3, and IL-6, or native,semi-purified stem cell proliferation factor (SCPF). SCPF is a cytokinethat promotes the proliferation of primitive hematopoietic stem cellswhile maintaining their CD34 phenotype. The cells were grown in thepresence of these exogenous cytokines for a period of several months.CD34 cells grown in the presence of IL-3 and IL-6 differentiated and thecultures were lost. However, the culture supplemented with SCPF wereSCPF-dependent and continued to proliferate. The SCPF-dependency waslost over time and the cell cultures became SCPF-independent. Thispopulation of cells was examined by flow cytometry and was shown toexpress CD34. This cell line was designated ML-1. The ML-1 cell line wascloned by limiting dilution and rescreened by flow cytometry and a subclone of ML-1 was established that was phenotypically more primitivethan that of the parental cell line. This cell line was designatedNSC1.1. Given the primitive nature of this cell population, this cloneof ML-1 was used as the immunogen for the hybridoma program.

[0049] The MG1-ligand is expressed as a cell-surface antigen on theNSC1.1 cell line. The antigen can be immunoprecipitated from extracts ofthis cell line as a glycosylated protein of approximately 186±5% kD(kilodalton) apparent molecular weight.

[0050] Antibodies that specifically label a subset of hematopoieticprogenitors are extremely useful in hematopoietic research because theyallow the isolation of relatively pure populations of immaturehematopoietic cells in a single step. Cells recovered with MG1 antibodycould be an appropriate normal cell population to compare with leukemicblast cells and to use in studies on the mechanisms of action of cells,factors, and genes that regulate hematopoietic cell proliferation anddifferentiation.

[0051] MG1 antibody did not recognize cells from three human leukemiccell lines (e.g., KG-1a, K562, HEL 92.1.7), and did not bind to humanperipheral blood cells. MG1 did not bind to mouse bone marrow cells.

[0052] Monoclonal anti-stem cell antibodies can be produced readily byone skilled in the art. The general methodology for making monoclonalantibodies using hybridoma technology is now well known in the art. See,e.g., M. Schreier et al., Hybridoma Techniques (Cold Spring HarborLaboratory 1980); Hammerling et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier Biomedical Press 1981); Kennett et al., MonoclonalAntibodies (Plenum Press 1980). Immortal, antibody-secreting cell linescan also be produced by techniques other than fusion, such as directtransformation of B-lymphocytes with oncogenic DNA or EBV. Severalantigen sources can be used, if desired, to challenge the normalB-lymphocyte population that is later converted to an immortal cellline.

[0053] For example, the NSC1.1 cell line can be used as an immunogen tochallenge the mammal (e.g., mouse, rat, hamster, etc.) used as a sourcefor normal B-lymphocytes. The antigen-stimulated B-lymphocytes are thenharvested and fused to an immortal cell line or transformed into animmortal cell line by any appropriate technique. A preferred hybridomaproducing the monoclonal MG1 antibody is produced by challenging a mousewith the NSC1.1 cell line and fusing the recovered B-lymphocytes with animmortal SP2/0-Ag14 myeloma cell. Antibody-producing immortal cells canbe screened for anti-stem cell antibody production by selecting clonesthat are strongly reactive with the NSC1.1 cells, but not reactive withgranulocytes from a panel of human donors. Antibodies produced by cloneswhich show those properties can then be screened for the additionalproperties of anti-stem cell antibodies.

[0054] A mouse hybridoma producing monoclonal MG1 antibody was depositedwith the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, on Nov. 6, 1996, and assigned ATCC Accession No.HB12232.

[0055] The subject cell line has been deposited under conditions thatassure that access to the culture will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35USC 122. The deposit is available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0056] Further, the subject cell line deposit will be stored and madeavailable to the public in accord with the provisions of the BudapestTreaty for the Deposit of Microorganisms, i.e., it will be stored withall the care necessary to keep it viable and uncontaminated for a periodof at least five years after the most recent request for the furnishingof a sample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cell line. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

[0057] In a preferred embodiment, the present invention encompasses anymonoclonal antibody that recognizes the MG 1 antigen, i.e., the antigenrecognized by antibody from the hybridoma ATCC HB12232. In anotherpreferred embodiment, the present invention contemplates monoclonalantibodies that correspond to the monoclonal antibody produced by ATCCHB12232, and, in a particularly preferred embodiment, the ATCC HB12232antibody. One antibody corresponds to another antibody if they bothrecognize the same or overlapping antigen binding sites as demonstratedby, for example, a binding inhibition assay.

[0058] An alternative to the above method of producing monoclonalantibodies employs the MG1 antigen directly as an immunogen. Themonoclonal antibody produced by hybridoma ATCC HB12232 can be readilyemployed to purify the MG1 antigen. In one example ofimmunopurification, MG1 antigen can be immunoprecipitated from cellextracts of the NSC1.1 cell line. The precipitated antigen can be usedas an immunogen in place of the NSC1.1 cell line in the above method. Byapplication of any of the above methods, one skilled in the art canreadily produce a panel of monoclonal antibodies, screen them withpartially purified MG1, and obtain anti-MG1-ligand antibodies.

[0059] Another alternative is to use an MG1 antibody in the productionof monoclonal antibodies that recognize different antigens on MG1⁺cells. The cells isolated from blood and marrow with MG1 antibody can beused as an immunogen, as described above, to produce a panel ofmonoclonal antibodies against MG1⁺ cells. The production of suchantibodies is greatly facilitated by the use of substantially purepopulations of hematopoietic cells provided by the MG1 antibody. Thespecificities of such antibodies can be determined a readily throughroutine screening by one skilled in the art. Thus, additionalstage-specific, a lineage independent antigens (and antibodies to theseantigens) can be identified by those skilled in the art.

[0060] MG1 recognizes a marker on the surface of hematopoietic cellsthat is distinct from CD34. Consequently, use of the MG1 antibodyprovides an alternative (or additional) means for the positive selectionof a subset of this population from human bone marrow or peripheralblood. Given that MG1⁺ cell populations represent only a subset of theCD34 population, antibodies immunoreactive with the MG1 antigen can beused in the field of tumor cell purging of bone marrow or peripheralblood stem cells, as well as in the preparation of stem cell populationsfor genetic therapy.

[0061] The antibodies according to the subject invention may be eithermonoclonal, polyclonal, or a mixture of monoclonal and/or polyclonalantibodies. The antibody may comprise whole antibody or antigen-bindingfragments thereof, such as Fab₂, Fab and Fv fragments. Antigen bindingfragments can be prepared using conventional techniques known in theart, such as proteolytic digestion of antibody by papain or pepsin, orthrough standard genetic engineering techniques known in the art.Monoclonal antibodies exemplified herein can be engineered so as tochange the isotype of the antibody. For example, the MG1 antibody, whichis an IgG_(2A) isotype, can be engineered as an IgG₁, IgG_(2B), or otherisotypes. Also contemplated by the subject invention are antibodies thatare reactive with the MG1 antibody and which have been engineered tocomprise human antibody constant regions. “Humanized” antibodies can beprepared using standard methods known in the art. See, for example, U.S.Pat. No. 5,585,089 (issued Dec. 17, 1996), the disclosure of which ishereby incorporated by reference.

[0062] The antibodies of the subject invention can be labeled accordingto standard methods known in the art. For example, antibodies can belabeled with detectable labels such as fluorescein, rhodamine andradioactive isotopes.

[0063] As indicated above, one application for monoclonal antibodies tolineage independent antigens on stem cells is the isolation of a highlyenriched source of stem cells for human bone marrow transplantation.Such sources of stem cells can prevent or attenuate Graft Versus HostDisease. Anti-stem cell monoclonal antibodies can also be used toisolate stem cells for autologous reinfusion, for example, in thetreatment of antigen-negative leukemias or other malignancies.

[0064] The present invention contemplates the use of any methodemploying the MG1 monoclonal antibody to separate stem cells from maturelymphocytes in the marrow or blood. Generally, a cell suspensionprepared from human tissue containing cells (i.e., marrow or bloodcells) is brought into contact with the MG1 monoclonal antibody. Cellsthat have been bound by the monoclonal antibody are then separated fromunbound cells by any means known to those skilled in the art.

[0065] Various methods of separating antibody-bound cells from unboundcells are known. For example, the antibody bound to the cell (or ananti-isotype antibody) can be labeled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in the art. In onepreferred embodiment, the anti-stem cell antibody is attached to a solidsupport. Various solid supports are known to those of skill in the art,including, but not limited to, agarose beads, polystyrene beads, hollowfiber membranes, polymers, and plastic petri dishes. Cells that arebound by the antibody can be removed from the cell suspension by simplyphysically separating the solid support from the cell suspension.Preferred protocols, however, will be described.

[0066] Selective cytophoresis can be used to produce a cell suspensionfrom human bone marrow or blood containing pluripotent hematopoieticstem cells. For example, marrow can be harvested from a donor (thepatient in the case of an autologous transplant; a donor in the case ofan allogeneic transplant) by any appropriate means. The marrow can beprocessed as desired, depending mainly upon the use intended for therecovered cells. The suspension of marrow cells is allowed to physicallycontact, for example, a solid phase-linked monoclonal antibody thatrecognizes an antigen on the desired cells. The solid phase-linking cancomprise, for instance, adsorbing the antibodies to a plastic,nitrocellulose, or other surface. The antibodies can also be adsorbed onto the walls of the large pores (sufficiently large to permitflow-through of cells) of a hollow fiber membrane. Alternatively, theantibodies can be covalently linked to a surface or bead, such asPharmacia Sepharose 6MB macrobeads. The exact conditions and duration ofincubation for the solid phase-linked antibodies with the marrow cellsuspension will depend upon several factors specific to the systememployed. The selection of appropriate conditions, however, is wellwithin the skill of the art.

[0067] The unbound cells are then eluted or washed away with physiologicbuffer after allowing sufficient time for the stem cells to be bound.The unbound marrow cells can be recovered and used for other purposes ordiscarded after appropriate testing has been done to ensure that thedesired separation had been achieved. The bound cells are then separatedfrom the solid phase by any appropriate method, depending mainly uponthe nature of the solid phase and the antibody. For example, bound cellscan be eluted from a plastic petri dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting a enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

[0068] The eluted, enriched fraction of cells may then be washed with abuffer by centrifugation and either cryopreserved in a viable state forlater use according to conventional technology or immediately infusedintravenously into the transplant recipient.

[0069] In a particularly preferred embodiment, stem cells can berecovered directly from blood using essentially the above methodology.For example, blood can be withdrawn directly from the circulatory systemof a donor and percolated continuously through a device (e.g., a column)containing the solid phase-linked monoclonal antibody to stem cells andthe stem cell-depleted blood can be returned immediately to the donor'scirculatory system using, for example, a conventional hemapheresismachine. When a sufficient volume of blood has been processed to allowthe desired number of stem cells to bind to the column, the patient isdisconnected from the machine. Such a method is extremely desirablebecause it allows rare peripheral blood stem cells to be harvested froma very large volume of blood, sparing the donor the expense and pain ofharvesting bone marrow and the associated risks of anesthesia,analgesia, blood transfusion, and infection.

[0070] The above cell populations containing MG1⁺ enriched humanhematopoietic cells can be used in therapeutic methods such as stem celltransplantation, as well as other methods that are readily apparent tothose skilled in the art. For example, such cell populations can beintravenously administered to a patient requiring a bone marrowtransplant in an amount sufficient to reconstitute the patient'shematopoietic and immune system. Precise, effective quantities can bereadily determined by those skilled in the art and will depend, ofcourse, upon the exact condition being treated by the therapy. In manyapplications, however, an amount containing approximately the samenumber of stem cells found in one-half to one liter of aspirated marrowshould be adequate.

[0071] In another embodiment, the MG1 monoclonal antibody can be used toisolate MG1⁺ cells, which can be used in various protocols of genetictherapy.

[0072] The optimal choice of target tissue for gene therapy is along-lived, preferably self-renewing cell. The hematopoietic stem cellis particularly attractive as a target for gene therapy for severalreasons. First, the procedures for the collection, cryopreservation, andreinfusion of human bone marrow are well developed, and the efficacywell established. Second, the use of stem cells or very earlypluripotential precursor cells would assure long term maintenance of thegenetically modified cells, and thus reduce the number of interventionsrequired. Third, one of the obstacles faced by gene therapists is thatsustained high level expression of transgenes has been difficult toachieve in large outbred mammals (Blaese, R. M., Clinical Immunology andImmunopathology, (61):547-555 (1991); Miller, A. D., Nature 357:455-460(1992)). One of the ways to address this problem has been to useexpression systems adapted to the target tissue (Kay et al., Hum. GeneTher. 3:641-647 (1992)). In the hematopoietic system, precursor (stem)cells can differentiate along one of three developmental pathways thatproduce large numbers of terminally differentiated cells, myeloid,lymphoid and erythroid cells. The control of gene expression during thedevelopment of the hematopoietic system has been extensively studied(Evans et al., Ann. Rev. Cell Biol. 6:95-124 (1990)), and elementsimplicated in the tissue-specific expression of genes have beenidentified for all three developmental pathways. The therapeutictransgene could be genetically modified to be constitutively expressedor expressed specifically in one of the differentiated hematopoieticlineages. Fourth, small numbers of hematopoietic stem cells produce verylarge numbers of differentiated cells; this diminishes the burden on thetransducing procedure to be of very high efficiency or throughput sincea small population of genetically modified stem cells will generate alarge population of genetically modified cells within the patient.Finally, since small numbers of genetically modified cells arenecessary, the risk associated with the introduction of large numbers ofgenetically modified cells into patients is also diminished.

[0073] The use of a stem cell-specific antibody need not be limited tothe purification of stem cells prior to a transfection procedure. Withthe goal of generating vectors for in vivo gene therapy, it has beenproposed to engineer into the gene therapy vectors themselves,mechanisms by which the vector will recognize its target cell (andpreferably only its target) within the context of the entire organism.See, Kasahara et al., Science 266:1373-1376 (1994); Michael & Curiel,Gene Therapy 1:223-232 (1994); Chatterjee et al., Ann. N.Y. Acad. Sci.770:79-90 (1995); Schwarzenberger et al., Blood 87:472-478 (1996). Byincorporating stem cell-specific antibodies into a vector, it may bepossible to generate vectors that will recognize and targethematopoietic stem cells in the patient's bone marrow. Specifically, theantibody could be incorporated into liposome vectors, (Hughes et al.,Cancer Res. 49:6214-6220 (1989); Wang & Huang, Biochemistry 28:9508-9514(1989); Ahmad et al., Cancer Res. 53:1484-1488 (1993)), poly-L lysineconjugate vectors (Michael & Curiel, supra; Schwarzenberger et al.,supra), or into viral vectors, including but not limited to adenoviralvectors, retroviral vectors, (Russell et al., Nucleic Acids Res.21:1081-1085 (1993); Somia et al., Proc. Natl. Acad. Sci. USA92:7570-7574 (1995)), and adeno-associated vectors (Chatterjee et al.,supra), modified to express on the vector surface, the antibody itselfor proteins which would bind the antibody to the vector surface (such asthe Fc receptor).

[0074] These vectors, although partly conceived for use in in vivo genetherapy can also be used to target the same MG1⁺ cells in ex vivoapplications, on either pre-selected MG1⁺ cells, or on whole bonemarrow, mobilized peripheral blood stem cells, or cord blood stem cells.

[0075] In a preferred method of in vivo gene therapy, genetic disorders,such as, e.g., sickle cell anemia, β-thalassemia, Fanconi anemia, andother hemoglobinopathies, may be corrected by the introduction of thenormal gene into a human stem cell, which can then be transplanted intoa patient's bone marrow. The treatment of genetic disease usinggenetically modified stem cells is not limited to the treatment ofhematopoietic tissues and diseases, but can also be extended to diseasesin which the presence of a circulating protein is of clinical benefit,such as Gaucher disease and hemophilia A and B.

[0076] To effect gene therapy with a substantially pure population ofhuman progenitor cells, the following method may be used to insert agene into these cells. For a general review of the methodologies, seeFriedmann, T., Science 244:1275-1281 (June 1989) and Lancet 1: 1271-1271(Jun. 4, 1988).

[0077] A therapeutic gene can be introduced into the population ofpurified stem cells, isolated as above by; (1) physical methods such ascoprecipitation with calcium phosphate, electroporation ormicroinjection (e.g., U.S. Pat. No. 4,873,191), and/or (2) the use ofviral vectors such as adenoviral, or retroviral vectors. In the lattercase, the DNA of the retrovirus is cut with a restriction enzyme and thehuman DNA containing the desired sequence is inserted and ligated. Theretrovirus containing the insertion is then infected into the stemcells. The stem cells can then be assayed for production of the desiredprotein. See, e.g., U.S. Pat. No. 4,902,783.

[0078] In general, molecular DNA cloning methods are well known in theart and are not limiting in the practice of this invention. For afurther description of similar methods, see Friedmann, T., Science244:1275-1281 (1989) and Molecular Cloning: A Laboratory Manual, 2nded., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0079] To transplant the stem cells containing the desired gene, thecells may be introduced into the bone marrow of the patient byconventional means of bone marrow transfer. Typically, this involves theintravenous delivery of the cells over a period of time. The bone marrowof the patient may be lethally irradiated prior to infusion to assurethat the transplanted stem cells fully replace the existing bone marrowcells. See U.S. Pat. No. 4,721,096 to Naughton et al. (Jan. 26, 1988).

[0080] In another method of gene therapy, drug resistance genes (i.e.,dihydrofolate reductase (dhfr), MDR1) can be introduced into normal stemcells and then transplanted into a cancer patient undergoingchemotherapy to protect the healthy stem cells from the toxic effects ofthe drug. This procedure would allow more aggressive chemotherapeuticregimens to be employed. (Guigon et al., Bone Marrow Transplant 13:93-95(1994); Baum et al, J. Virol. 69:7541-7547 (1995)). MG1⁺ cellpopulations may be useful in this method. Modified stem cells also haveapplications in the treatment of certain acquired diseases such as HIVinfections (Nienhuis et al., Cancer 67(10 Suppl.):2700-2704 (1991);Bahner et al., J. Virol. 70:4352-4360 (1996)).

[0081] In another method of gene therapy, the MG1 antibody can be usedin an antibody modified delivery system to target drug deliveryspecifically to MG1⁺ hematopoietic cells in vivo. See, e.g., Ahmad etal., Cancer Res. 53:1484-1488 (1993).

[0082] The subject invention also pertains to the gene encoding the MG1antigen polypeptide. Contemplated within the scope of the invention arenatural and allelic variant polynucleotide sequences encoding the MG1antigen, as well as degenerate polynucleotide sequences that encode theMG1 antigen polypeptide. Polynucleotide sequences encoding the MG1antigen can be readily obtained using materials of the subject inventionand standard methods known in the art. For example, a cDNA library canbe prepared from NSC1.1 cells and that cDNA library inserted into anappropriate expression system, such as lambda phage. Clones can then bescreened for expression products using, for example, an MG1 antibody.Clones that are positive for expression of a polypeptide that binds toMG1 antibody can be further evaluated by sequencing of the cDNA insert,Western blot, etc.

[0083] Polynucleotide sequences encoding the MG1 antigen can also beobtained using degenerate oligonucleotide probes based on the amino acidsequence of the MG1 antigen in conjunction with standard RACE proceduresknown in the art. For example, 5′ RACE can be performed using a MARATHONcDNA amplification kit (CLONTECH Laboratories, Palo Alto, Calif.) toamplify a 5′ RACE fragment from polyA⁺ RNA obtained from cells thatexpress the MG1 antigen. The RACE fragments generated using thedegenerate oligonucleotide probes can then be cloned and characterizedby sequencing. Full length cDNA sequences can then be generated by endto end PCR or by conventional cloning methods known in the art.

[0084] Other methods for screening for polynucleotide sequences encodingthe MG 1 antigen are known in the art. These include, for example,screening DNA libraries using degenerate oligonucleotide probes that canbe prepared based on the partial amino acid sequence of the MG1 antigen.Also contemplated within the scope of the present invention arefragments and variants of the polynucleotide encoding MG1 antigen, aswell as fragments and variants of the MG1 antigen itself. The fragmentsof the subject polynucleotides can be readily prepared using standardmethods known in the art. For example, digestion using the BAL31exonuclease can be used to prepare 5′ and 3′ nucleotide deletions.

[0085] The subject invention also pertains to anti-idiotypic antibodiesthat possess binding specificity for idiotypic determinants associatedwith anti-MG1 antigen antibodies of the present invention. Theanti-idiotypic antibodies that the invention can be prepared using theanti-MG1 antigen antibodies as an immunogen according to standardmethods for producing anti-idiotypic antibodies known in the art. Alsoincluded within the scope of the present invention are antigen bindingfragments of whole anti-idiotypic antibody, wherein the fragments retainsubstantial by the same binding specificity as the whole antibodymolecule. The antigen binding fragments include, for example, Fab₂, Faband Fv fragments.

[0086] The subject invention also concerns kits comprising a compartmentcontaining at least one anti-MG1 antigen antibody, anti-idiotypicantibody or an MG1 antigen. In one embodiment, the anti-MG1 antigenantibody is the MG1 antibody disclosed herein.

[0087] Antibodies to the MG1 antigen can also be used to investigatedifferential expression of the MG1 antigen on tumor cells versus normalcells. Thus, the subject invention also concerns methods for identifyingtumor cells, such as leukemic cells, from normal cells by contacting asample with an antibody of the invention and determining whether theantibody binds to any of the test cells. In a preferred embodiment, MG1antigen expression on cells is determined using the MG1 antibody of thepresent invention.

[0088] The subject invention also concerns methods for treating tumorsin patients using an antibody of the subject invention that binds to anMG1 antigen. In a preferred embodiment, the method comprisesadministering an effective amount of an MG1 antigen binding antibody toa patient in need of such treatment. Methods for administeringantibodies to treat various disease states are known in the art.Preferably, the antibody is modified in a manner so as to minimize anyimmune response to the antibody when it is administered to the patient.For example, the antibody can be “humanized,” such as by replacingnon-human constant regions of the antibody with human constant regions,according to methods known in the art. A variety of toxic agents capableof killing or inhibiting the replication of a cell can be conjugated tothe antibody. For example, a variety of cytotoxic agents are availablein the art. These include, for example, radionuclides (Iodine-13 1,Yttrium-90 and the like), chemotherapeutic agents (such as methotrexate,cisplatinum and the like) and cytotoxic proteins (such as ricin,exotoxins, diphtheria toxins and the like). In one embodiment, themethod can be used to treat leukemia.

[0089] The following examples are provided to illustrate specificembodiments of the present invention. The examples are included forillustrative purposes only, and are not intended to limit the scope ofthe present invention.

Materials and Methods

[0090] Preparation of Cell Line for Use as an Immunogen

[0091] NSC1.1 cells were grown in T-75 cm² tissue culture flasks usingthe following growth medium: Iscove's modified Dulbecco's medium (IMDM)supplemented with 10% fetal bovine serum (FBS), L glutamine (0.292mg/ml) and antibiotics (50 Units/ml penicillin, and 50 Units/mlstreptomycin). Once the NSC1.1 cells reached confluency, thesupernatants were harvested, centrifuged (2000 rpm for 4 minutes at roomtemp.), and the cell pellet resuspended in IMDM growth medium (as statedabove). The concentration of viable cells at time of harvest wasestimated (using trypan blue dye exclusion) and the cell suspensionadjusted to a concentration of 1.5×10⁸ cells/ml. The cells were thenwashed four times in sterile phosphate buffered saline (PBS) at pH 7.5and finally resuspended in one ml of PBS at a concentration of1.5×10⁸/ml.

[0092] Mice Immunization Protocol

[0093] Three 23-27 day-old female Balb/c mice (Charles RiverLaboratories, Md.) were injected with 200 μl of the NSC 1.1 cellsuspension (3×10⁷ total cells) via the intra-peritoneal (I/P) route. Asa control, one mouse was injected, I/P, with 200 μl of PBS only.Following the initial injection, the mice were immunized in the mannerand over the time course set out below: Time Protocol Route Inoculum Day0 (1/23/95) Primary Immun. I/P 3 × 10⁷ M1-1 cells Day 15 (2/7/95) FirstBooster I/P 3 × 10⁷ M1-1 cells Day 23 (2/17/95) Second Booster I/P 3 ×10⁷ M1-1 cells Day 33 (2/27/95 Final Booster¹ I/V^(ii) 10⁷ M1-1 cellsDay 36 (3/2/96) Harvest spleen for fusion & hybridoma production

[0094] Established Cell Lines Used

[0095] Sp2/0-Ag14:

[0096] Sp2/0-Ag14 (ATCC CRL-1581) is a non-secreting myeloma hybrid ofmurine origin that is routinely used in the fusion process of hybridomaproduction.

[0097] KG1a:

[0098] KG1a (ATCC CCL-246.1) is a variant subline of the human, acutemyelogenous leukemia cell line KG1 (ATCC CCL-246). KG1a was the cellline that was used to manufacture the MY10 monoclonal antibody thatdefines the CD34 antigen. See U.S. Pat. No. 4,965,204 (Oct. 25, 1990) toCivin.

[0099] K562:

[0100] K562 (ATCC CCL-243) is a continuous cell line established from ahuman with chronic myelogenous leukemia. The cell line is characterizedas a highly undifferentiated blast of the granulocytic series. Recentstudies indicate that the K562 cells are undifferentiated blasts thatare multipotential and capable of differentiating into progenitors ofthe erythrocytic, granulocytic, and monocytic series.

[0101] HT-29:

[0102] HT-29 (ATCC HTB-38) is a human colon adenocarcinoma.

[0103] HEL 92.1.7: Hel 92.1.7 (ATCC TIB-180) is a lymphoblastic-likecell line derived from an erythroleukemia. This cell line is capable ofboth spontaneous and induced globin synthesis.

[0104] Hybridoma Production

[0105] Prior to the fusion, Sp2/0-Ag14 myeloma cells were tested fortheir sensitivity to Hypoxanthine, Aminopterin, Thymidine (HAT)containing medium. The Sp2/0-Ag14 myeloma cells are hypoxanthine-guaninephosphoribosyl-transferase (HGPRT) negative and, therefore, aresensitive to the presence of HAT whereas normal spleen cells are HGPRT⁺and are resistant to HAT. The HGPRT mutation was, therefore, used in thepositive selection of hybrid cells (spleen/myeloma).

[0106] HAT-sensitive Sp2/0-Ag14 cells were harvested, from culture,centrifuged (2000 rpm, 4 minutes), and the pellets resuspended in IMDMsupplemented with L glutamine (0.292mg/ml) and antibiotics (50 Units/mlpenicillin, and 50 Units/ml streptomycin). Cell counts were performed bytrypan blue dye exclusion.

[0107] The 3 experimental mice (immunized with NSC1.1 cells as describedabove) were sacrificed by cervical dislocation and their spleens removedusing aseptic techniques. The spleens were kept separate and placed in100 mm petri dishes containing 10 ml of the IMDM supplemented with Lglutamine (0.292 mg/ml) and antibiotics (50 Units/ml penicillin, and 50Units/ml streptomycin). Cell suspensions from each spleen were made inthe above medium, washed twice and viable cells enumerated, by trypanblue dye exclusion. The Sp2/0-Ag14 myeloma cell and spleen cellsuspension were mixed together at a ratio of 1:5 (myeloma:spleen) in thepresence of polyethylene glycol (PEG) 1500 (Boehringer Mannheim).

[0108] The fusion mixture was then incubated at 37° C. for 10 minutes.Following the incubation step, the cell-fusion mixture was centrifugedand resuspended in 30 ml of fresh IMDM supplemented with 20% fetalbovine serum (FBS), L glutamine (0.292 mg/ml) and antibiotics (50Units/ml penicillin, and 50 Units/ml streptomycin). The myeloma/spleencell fusion mixture, for each mouse, was added in 100 μl volumes (5×10⁵spleen cells/well) to each well of three 96-well microplates. Themicroplates (a total of 9 plates) were then incubated at 37 EC in ahumidified atmosphere of 5% CO₂ in air. Four hours after incubation at37° C., 100 μl of IMDM containing 20% fetal bovine serum (FBS), Lglutamine (0.292 mg/ml) and antibiotics (50 Units/ml penicillin, and 50Units/ml streptomycin) and further supplemented with 2×HAT was added toeach well of the nine 96-well microplates.

[0109] All the microplates were then re-incubated at 37° C. in ahumidified atmosphere of 5% CO₂ in air and checked daily for theappearance of hybridoma growth. Every third day, 100 μl of supernatantfrom each of the wells was removed and replenished with fresh IMDMgrowth media plus HAT. The media removed from the individual wells wasthen used to screen for positive NSC1.1 reactivity. Screening wascarried out using a sandwich ELISA and/or immunocytochemistry.

[0110] The multi-clone wells that were identified as having NSC1.1specific immunoglobulins in the supernatant, by either test, were grownto high cell density and subsequently passaged into new wells of a96-well microplate. These multi-clones were routinely screened forreactivity with the immunizing NSC1.1 cell line. If the clone(s)maintained their reactivity they were expanded to tissue culturecontainers with greater surface area. Excess cells from each positiveclone were stored in liquid nitrogen. Once positive clones wereestablished, the antibody reactivity was tested (ELISA, flow cytometry,and/or immunocytochemistry) against a wider variety of targets, whichincluded cell lines, KG1a, K562, HT-29, and HEL 92.1.7, described above,and peripheral blood leukocytes (granulocytes, lymphocytes, monocytes,and thrombocytes). All clones with unique antibody profiles were thensubjected to limiting dilution cloning to produce a monoclonal hybridomasecreting an antibody of a single isotype and with a defined antigenspecificity.

[0111] Detection of Antibody-Positive Clones by a Sandwich-Enzyme LinkedImmunoadsorbent Assay (ELISA)

[0112] The sandwich ELISA technique used was modified from the method asdescribed by Harlow & Lane, “Chapter 14: Immunoassays,” in Antibodies: ALaboratory Manual, Harlow & Lane, eds., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988), pp. 553-612. Briefly, the ELISAwas run in U-shaped, 96-well microplates whose wells had been previouslyblocked, overnight, with 3% bovine serum albumin (BSA) in PBS, (pH7.5).Cells to be used as the antigen (NSC1.1, KG1a, K562, HT29, andHEL92.1.7) were washed three times in PBS, resuspended in PBS,enumerated (by trypan blue dye exclusion) and the cell number adjustedto 2×10⁶/ml. One hundred microliters (2×10⁵ cells/well) of these cellsuspensions were then added to each well of 96-well plates. All theplates containing the cells were subjected to centrifugation (2000 rpm,for 5 minutes at 4° C.) and the supernatants from each of the wellsaspirated. Cells were resuspended in 100 μl of undiluted test hybridomasupernatant and incubated at 28° C. (room temperature) for 60 minutes.Each test hybridoma was run in triplicate wells. Following incubationwith the hybridoma supernatant, the cells were centrifuged (2000 rpm, 4°C., for 4 minutes) washed once with PBS and the cell pellets resuspendedin 50 μl of biotinylated rabbit/goat anti-mouse immunoglobulin (Ig)(Vectastain Elite ABC Kit, Vecta Labs, Burligame, Calif.). The plateswere then re-incubated at 28° C. for a further 60 minutes. The cellswere again washed once in PBS and then resuspended in 100 μl ofVectastain ABC Avidin-HRP conjugate with a final incubation of 30minutes at 28° C. After incubation, the cells were washed once with PBSand the pellet resuspended in 100 μl of substrate-2,2 Azino-bis-3ethylbenz-thiazoline-6 sulphonic acid (ABT) (Sigma Chemicals Co, St.Louis, Mo.). The reaction was allowed to proceed for ten minutes, afterwhich the reaction was stopped. The assay was readspectrophotometrically at 405 nm and the data recorded. Results weredetermined to be positive if the O.D. reading was 2 standard deviationsabove the negative control.

[0113] Detection of Antibody-Positive Clones by Immunocytochemistry

[0114] The immunocytochemical procedure was as described in themanufacturer's instructions for the use of the Vectastain ABC Elite Kit(Vecta Labs, Burligame, Calif.). Briefly, cells to be used (NSC1.1,KG1a, K562, HT29, HEL 92.1.7) were centrifuged (2000 rpm, 4° C., for 4minutes) and the cells resuspended in PBS. The cells were washed afurther 5 times in PBS and finally resuspended, in PBS, enumerated(Trypan blue dye exclusion) and the cell concentration adjusted to1×10⁶/ml. Two hundred microliters of each cell suspension were added tothe cytospin chambers of a cytocentrifuge and were then centrifuged ontoglass slides (500 rpm for 5 minutes). Once the cells were deposited ontothe glass microscope slides, they were air-dried then fixed in methanolat room temperature. Prior to testing hybridoma supernatants, theendogenous peroxide activity was quenched by incubating the cell smearswith a 1% solution of hydrogen peroxide (H202) solution for one hour atroom temperature. The slides were washed of excess H202 and thenincubated for 60 minutes at room temperature with the undiluted testhybridoma supernatants. Following this step, the excess supernatant wasremoved, the slides rinsed in 3×250 ml beakers containing fresh PBS, andeach of the cell smears incubated at room temperature for 30 minuteswith 50 μl biotinylated rabbit/goat anti-mouse Ig. Following the secondantibody step, the slides were again washed in PBS, as stated above, andincubated at room temperature for 30 minutes with 50 μl of Avidin-HRPconjugate, washed, then incubated for ten minutes with 100 μl of thesubstrate 3,3 diaminobenzidine (DAB). The reaction was stopped, airdried and the cells permanently fixed and mounted under Dpex and a glasscoverslip. All slides were then examined by light microscopy (usingeither 40× objective or 40× oil objective). The results were scored asfollows: ++++ Very strong positive reaction +++ Strong positive reaction++ Positive reaction + Weak positive reaction − Negative reaction

[0115] Detection of Antibody Positive Clones by Flow Cytometry

[0116] For the detection of binding of specific antibody to the surfaceof NSC1.1 cells, flow cytometry was carried out as described below.Briefly, staining of the NSC1.1 cells was performed on two-to-three dayold cell cultures. Cells were adjusted to a cell concentration of 1×10⁶cells per sample (200 μl) in IMDM containing 1% fetal bovine serum(FBS), L glutamine (0.292 mg/ml) and antibiotics (50 Units/mlpenicillin, and 50 Units/ml streptomycin). To the cells was added 100 μlof the test hybridoma supernatant, the mixture vortexed, and incubatedfor 30 minutes on ice. Following incubation, the samples were washed inthe above media and the cells resuspended in 200 μl of IMDM medium(shown above) and 50 μl of fluorescein isothiocyanate (FITC)-labeledgoat anti-mouse IgG (Fab)′2 (GAM-FITC). The labeled cells were thenincubated for 30 minutes on ice. After labeling, the cells were againwashed, resuspended in 1 ml of the above IMDM medium and then subjectedto flow analysis using an Epics Elite ESP (Coulter Corporation, Hialeah,Fla.) equipped with a 488 nm argon air-cooled laser. Forward and sidescatter gates were adjusted to include live cells only. Results of theanalysis were based on the collection of 10,000 data points per sample.

[0117] Separation of Peripheral Blood Leukocyte Sub-Sets by SelfGenerated Percoll Gradients

[0118] Subsets of peripheral blood leukocytes were separated usingPencoll (Pharmacia, Piscataway, N.J.) according to the method describedin the manufacturer's instructions. The self generating 70% Percoll (in0.1 5M NaCl) gradients were generated by centrifuging 10 ml of Percollsolution in 15 ml pyrex glass tubes (Corning, Cambridge Mass.), at20,000×g (J20 rotor) for 15 minutes at 10° C. Two ml of fresh citrated(an anticoagulant) peripheral blood was then overlaid onto the Percollgradients and centrifuged at 800×g for 25 minutes at 10° C. Followingcentrifugation, the blood cells were separated according to theirdensities, i.e., platelets remained at the blood/Percoll interface,mononuclear cells (lymphocytes and monocytes) banded in the center ofthe gradient, and the polymophonuclear cells (granulocytes) anderythrocytes banded to the bottom of the gradients. Using a 5 ml pipettethe layers of discrete cells were harvested from the gradient, placed inseparate 15 ml polypropylene tubes, centrifuged (2000 rpm for 4 minutesat room temperature) and the pellets washed 3 times in IMDM (withoutsupplements). The final cell pellet was then resuspended in IMDM(without supplements) at the required cell concentrations for eitherimmuno-staining or western blot analysis.

[0119] Characterization of the Monoclonal Antibody Isotype

[0120] The identification of the monoclonal antibody isotype(s), beingsecreted by hybridomas of interest, was carried out using a mouse Igtyping kit (Pharmingen, San Diego, Calif.). The procedure was asdescribed in the manufacturer's instructions for use. Briefly, 100 μl ofrat monoclonals, with specificity for each mouse Ig isotype wasaliquoted in wells (8 wells/isotype) of a flat-bottomed microplate andincubated at 4° C. overnight. Following the overnight incubation, thesupernatants from each well were aspirated, 300 μl of a 3% BSA solution(blocking agent) was added, and the plates incubated for a further 30minutes at room temperature. Following this incubation step, themicroplates were washed 5 times with PBS/TWEEN 20 (0.5%) and 100 μl oftest hybridoma supernatant added after the last wash. The plates wereincubated for 60 minutes at room temperature. After incubation, theplates were then washed, as previously described, and 100 μl of alkalinephosphatase labeled polyclonal rat anti-mouse Ig added, and the platesre-incubated for 60 minutes at room temperature. Prior to the substratebeing added, the plates were washed 5 times in PBS/TWEEN 20. Thesubstrate (p-N.P.) and its concentration used were as suggested in theisotyping kit. Following the color development, the results weredetermined using a spectrophotometer at a wave length of 405 nm.

[0121] Preparation of Cell Lysates for SDS-PAGE

[0122] Cells were harvested, centrifuged (2000 rpm at room temperature)and washed 4 times in PBS. After the final wash, the cell pellets wereresuspended (vortexed) in lysis buffer (0.1% TRITON-X 100 in PBS, and 1mM phenylmethyl-sulfonylfluoride (PMSF) and incubated on ice for 60minutes. After incubation, the cell lysates were clarified bycentrifugation (14,000 rpm at 4° C. for 45 minutes) and the supernatantcollected and used as a stock cell lysate preparation.

[0123] Protein Molecular Weight Determination by Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDS-PAGE)

[0124] The SDS-PAGE procedure used was as described by Laemmli, Nature227:680-685 (1970). Samples (cell lysates) were prepared for onedimensional SDS-PAGE by boiling the lysate for 5 minutes in the presenceof SDS and 2.5% β-mercaptoethanol. Protein samples were routinelyresolved using a premade 10% polyacrylamide gel (BioRad Inc., Richmond,Calif.). Gels were run at a constant voltage of 75V for approximately4-5 hours. Pre-stained SDS-PAGE molecular weight standards (BioRad Inc.,Richmond, Calif.) were routinely included on each gel. Electrophoresedgels were stained either using Coomassie blue-R250 (Sigma Chemicals, St.Louis, Mo.) or by silver stain (BioRad Inc., Richmond, Calif.). Theapproximate molecular weight of proteins of interest was calculatedusing regression analysis.

[0125] Detection of Antibody Ligands by Western Blot Analysis

[0126] Western blot analysis using the ECL detection system (Amersham,Arlington Heights, Ill.) was carried out as described by Boman et al.,Nature 358:512-514 (1992); Dalemans et al., Nature 354:526-528 (1991);Egan et al., Nature 358:581-584 (1992); and Kleijmeer et al., Nature357:342-344 (1992). Cell associated proteins were separated usingSDS-PAGE, as previously described. These SDS-PAGE gels were thenelectroblotted onto pre-blocked nitrocellulose membranes (BioRad Inc.,Richmond, Calif.). The blocking of nitrocellulose membranes wasaccomplished by overnight treatment at 4° C. of the membranes with 5%non fat dry skimmed milk powder dissolved in PBS plus 0.01% TWEEN 20.The electroblot and transfer of the proteins to the nitrocellulose wasperformed using a Tris-Glycine transfer buffer containing 0.1% SDS andwas carried out for 90 minutes at a constant voltage (100V). Followingtransfer, the nitrocellulose membranes were washed in a wash buffer(PBS, pH7.5, plus 0.01% Tween20) and then incubated at room temperaturefor 60 minutes with primary antibody (1/10 dilution of hybridomasupernatant in 25 ml of blocking solution or 14 μg of purified antibodyin blocking solution). Following the incubation with the primaryantibody the membranes were washed in PBS/0.01% Tween20 and thehorseradish peroxidase labeled goat anti-mouse IgG secondary antibodywas added (1/2000 dilution). The membranes were re-incubated at roomtemperature for 30 minutes then washed three times in PBS. Detection ofantigen/antibody complexes was performed using the chemiluminescentreagent from Amersham's ECL system. The resulting X-ray films weredeveloped using a Kodak M35A X-OMAT processor and the autroradiographsexamined for positive reactions.

[0127] Determination of Protein Glycosylation

[0128] The presence/absence of carbohydrate moieties on the MG1 antigenwas assessed by enzyme deglycosylation of the protein backbone. In thisassay, digestion of any sugar present results in a shift in molecularweight that can be visualized by SDS-PAGE and western blot analysis.Deglycosylation of the MG1 antigen was carried out using the followingenzymes: for O-linked sugar determination, O-glycosidase (BoehringerMannheim, Indianapolis, Ind.), for N-linked sugar determination,N-glycosidase (Boehringer Mannheim, Indianapolis, Ind.). Briefly, NSC1.1lysate (50 μg total protein) was aliquoted into 1.5 ml microfuge tubesand to these tubes was added either N-glycosidase (6 Units/50 μgprotein), O-glycosidase (25 mUnits/50 μg protein) or a combination ofthe two. A microfuge tube containing 50 μg of protein only (no enzymes)was used as an untreated control. All of the tubes were incubated for 24hours in a 37° C. waterbath. Following incubation, 100 μl of 2×SDSreducing sample buffer was added to all tubes, and then placed in aboiling waterbath for 5 minutes. The samples were run on a SDS-PAGE (aspreviously described), electroblotted onto a nitrocellulose filter, andthen probed using the MG1 monoclonal antibody (as described above).

[0129] Purification of Mouse Monoclonal Immunoglobulins by AffinityChromatography

[0130] The purification of immunoglobulins from 3.2B11.b3 hybridomasupernatants, using affinity-based chromatography, was as described bySchwartz, L., “Use of immobilized protein A to purify immunoglobulins,”in Bacterial Immunoglobulin-Binding Proteins: Applications inImmunotechnology, M. D. P. Boyle, Academic Press (1990), pp. 309-339,and Walker, W. B., “Use of immobilized protein G to isolate IgG,” inBacterial Immunoglobulin-Binding Proteins: Applications inImmunotechnology, M. D. P. Boyle, Academic Press (1990), pp. 355-368.Briefly, stock cultures of the hybridoma were centrifuged (2000 rpm, for4 minutes at room temperature), then resuspended in fresh IMDMsupplemented with 5% FBS (Ultra-low IgG, Life Technologies), and placedin T₁₅₀ cm² tissue culture flasks and grown at 37° C., in a humidifiedatmosphere of 5% CO₂ in air. Once the hybridoma reached confluency,supernatants from the cultures were harvested, centrifuged (2000 rpm for10 minutes at room temperature), and then sterilized using a 0.22 μmfilter (Amicon, Beverly, Mass.). Five ml HiTrap protein G columns(Pharmacia Biotech, Piscataway, N.J.) were equilibrated with 20 ml of 20mM sodium phosphate buffer (pH 7.0). Approximately 500 ml of hybridomasupernatant was loaded using a peristaltic pump, onto eachpre-equilibrated column at the flow rate of 1 ml/min. The column wasthen placed in line on a Pharmacia Fast Protein Liquid Chromatography(FPLC) system and again washed with 20 mM sodium phosphate, buffer (pH7.0) until the UV detector/chart recorder returned to baseline. Thebound IgG was then eluted with 10-15 ml of 0.1M glycine buffer (pH 2.7)at a flow rate of 1 ml/minute and collected as a single fraction in atube containing 200 μl of 1M Tris-HCL (pH 9.0).

[0131] The purified IgG fraction was desalted against 20 mMsodium-phosphate buffer (pH 7.0) using PD-10 gel filtration columns(Pharmacia Biotech, Piscataway, N.J.) as per manufacturers instructions.Concentration of the purified IgG was carried out using a Centriplus 10(Amicon) and centrifuged at 3000×g for 60 minutes. Protein concentrationwas determined by Bradford protein assay and SDS-PAGE gels were run toascertain the purity of the immunoglobulin preparation. The antibody wasthen stored at 4° C. or -20° C.

[0132] Purification of MG1 Antigen by Affinity Chromatography

[0133] Purification of the cell surface antigen or ligand recognized bythe MG1 monoclonal antibody was carried out by immuno-affinitychromatography as previously described (Harlow & Lane, “Chapter 13:Immunoaffinity Purification,” in Antibodies: A Laboratory Manual, Harlow& Lane, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1988), pp. 511-552). Purified MG1 IgG was covalently coupled tothe sepharose matrix of a HiTrap NHS-activated 5 ml column (PharmaciaBiotech, Piscataway, N.J.) as per manufacturer's instructions. Briefly,the column was connected to a peristaltic pump and washed with 30 ml ofice-cold 1 mM HCL at a flow rate of 2 ml/min. Twelve mg purified MG1 IgG(in 25 ml of 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3) was then pumped onto thecolumn at the flow rate of 1 mmin. The MG1 containing solution wasrecirculated and pumped back over the column. The column was incubatedfor 30 minutes at room temperature (25° C.). Excess active groups weredeactivated by washing the column with 0.5 M ethanolamine, 0.5 M NaCl,pH 8.3 and non-specifically bound ligands were eluted off the columnwith 0.1 M acetate, 0.5 M NaCl, buffer (pH 4.0).

[0134] The MG1-immuno-affinity column was equilibrated with 30 ml of 20mM sodium-phosphate (pH 7.0). Ten ml of NSC1.1 cell lysate (prepared asstated above) was loaded into a 10 ml FPLC superloop (Pharmacia Biotech,Piscataway, N.J.) and injected onto the column at the flow rate of 0.5ml/min. The column was then washed with 20 mM sodium-phosphate, (pH 7.0)until no UV absorbance was recorded in the flow-through. The boundligands were eluted with 10 ml of 0.1M glycine (pH 2.7) and collected asa single fraction in a tube containing 200 μl of 1 M Tris-HCl, (pH 9.0).

[0135] The eluted fraction containing the MG1 antigen was desaltedagainst 20 mM sodium-phosphate buffer, (pH 7.0) using PD-10 gelfiltration columns (Pharmacia Biotech, Piscataway, N.J.) as permanufacturer's instructions. Concentration of the purified MG1 antigenwas carried out using a Centriplus 10 (Amicon) and centrifuged at 3000×gfor 60 minutes. Purity of the MG1 antigen and its apparent molecularmass was assessed by SDS-PAGE and Western blotting.

[0136] Determination of the Amino Acid Composition of the MG1-Antigen

[0137] The amino acid composition of the MG1-antigen was determined asfollows. The MG1-antigen was purified by immunoaffinity columnpurification, followed by SDS-PAGE and transfer of the ligand band toPVDF membranes. The amino acids were quantitatively released, withoutdegradation, by hydrolysing 0.45 μg of the purified MG1-antigen with 6NHCl for 24 hours at 110° C. under vacuum. After hydrolysis, the samplewas dried and reconstituted for analysis.

[0138] Amino acid analyses were performed on the Applied BiosystemsModel 420A Analyzer, an automated PTC amino acid analysis system.Briefly, the sample was applied to a glass frit support, which deliveredthe sample to a flow-through reaction chamber where the amino acids werederivatized with PTC using 5% phenylisothiocyanate in heptane. Afterderivatization, the PTC-amino acids were transferred on-line to an HPLCwhere each of the PTC-amino acids is separated based on its retention ona reverse phase C 18 HPLC column. Amino acid composition is determinedwith an overall error of approximately 10%, however, by restrictingfurther analyses to specific amino acids that give more reliableresults, it is possible to improve the accuracy. The amino acidcomposition data was used to attempt to identify the protein bycomparison to the available protein databases using the ExPASy,(Wilkins, M. R. et al., Bio/Technology 14:61-65 (1996), and Wilkins, M.R et al., Biochem. Biophys. Res. Commun. 221:609-613 (1996)), andPropsearch (Hobohm, U., et al., Analytical Biochemistry 222:202 (1994))programs.

[0139] Detection of Laminin Reactivity by Western Blot Analysis

[0140] The reactivities of the MG1 antibody against mouse laminin, andof the MG1-antigen with anti-laminin polyclonal antibodies, weredetermined by western blot, essentially as described above. Briefly,NSC1.1 lysate, NSC1.1 conditioned medium, and purified mouse laminin,were run in triplicate on SDS-PAGE and transferred to nitrocellulosemembranes as described. Following transfer, the nitrocellulose membraneswere separated into three parts and the individual parts were washed ina wash buffer (PBS, pH7.5, plus 0.01% TWEEN 20) and then incubated atroom temperature for 60 minutes with primary antibody. The membraneswere labeled with anti-laminin polyclonal antibody (rabbit serum,against human placental laminin, Chemicon International Inc.; lot #58296268) at a 1:2000 dilution, or MG1 monoclonal antibody (1/10dilution of hybridoma supernatant in 25 ml of blocking solution or 14 μgof purified antibody in blocking solution), or PBS alone. Followingincubation with the primary antibody, the membranes were washed inPBS/0.01% Tween20 and horseradish peroxidase labeled goat anti-mouse (ordonkey anti-rabbit) IgG secondary antibody was added (1/2000 dilution).The membranes were re-incubated at room temperature for 30 minutes, thenwashed three times in PBS. Detection of antigen/antibody complexes wasperformed using the chemiluminescent reagent from Amersham's ECL system.The resulting X-ray films were developed using a Kodak M35A X-OMATprocessor and the autroradiographs examined for positive reactions.

EXAMPLE 1 Characterization of the MG1 Monoclonal Antibody

[0141] The Derivation of the 3.2.B11.b.3 Hybridoma Subclone

[0142] The three mice immunized with the NSC1.1 subclone (NSC-1. 1) weresacrificed and their spleens used in fusion experiments for hybridomaproduction. Use of the spleens for fusion was based upon the reactivity,as measured by both flow cytometry and immunocytochemistry, of theindividual mouse sera with NSC1.1 cells. The sera from all three micegave very strong reactions using both detection systems.

[0143] The origin of the NSC1.1-specific hybridoma sub clone(3.2.B11.b3) is shown in FIG. 1. Due to bacterial contamination of themouse#2 spleen/SP2/0 fusion culture, no clones developed. From theremaining two mice (#1 and #3), 178 hybridomas (polyclones) wererecorded. Supernatants removed from all 178 hybridomas were testedutilizing an indirect ELISA. Thirty of these hybridomas gave strongpositive reactions against the NSC1.1 cell line. Over a period of twoweeks, the 30 hybridoma cultures were expanded and continually monitoredfor reactivity to the NSC1.1 cell line. During this period, eighthybridomas were lost. The ELISA screening of the remaining 22 clones wasalso expanded to include erythrocytes as an antigen. Clones that hadsignificant reactivity to both NSC1.1 cells and erythrocytes were thendiscarded. From these experiments, only 10 hybridoma clones gavepositive reactions against NSC1.1 cells alone. Following further passageof these 10 clones, 4 clones were lost. The remaining 6 clones wereexpanded to T75 cm² tissue culture flasks and samples from each storedin liquid nitrogen. Verification of NSC1.1 reactivity by all 6 cloneswas assessed by immunocytochemistry. Three clones with the strongestreactivity (3.2.B2, 3.2 B11, and 1.3.D9) were then sub cloned bylimiting dilution to obtain single cell hybridomas. All attempts to subclone 1.3 D9 to obtain single cell clones failed. Three monoclones weregenerated from the limited dilution cloning of 3.2.B2 and 7 monocloneswere generated from the limited dilution cloning of 3.2.B11, one ofwhich was 3.2.B11.b3. This clone was subsequently shown to secrete amonoclonal antibody of unique specificity and was designated “MG1.”

[0144] Purification and Isotype Identification of the MonoclonalAntibody Secreted by the Hybridoma 3.2.B11.b3 (MG1)

[0145] The monoclonal antibody secreted by the 3.2.B11.b3 hybridoma waspurified to homogeneity using protein-G affinity chromatography. Theprotein eluted from the column was subjected to SDS-PAGE electrophoresisto ascertain the purity of the preparation. The photomicrograph of aCoomasie blue-stained SDS-PAGE of the eluted protein, at differentconcentrations (FIG. 2), shows two distinct bands; one protein band atapproximately 60 kDa (an equivalent molecular weight to the heavy chainof an Ig) and the other at approximately 28 kDa (an equivalent molecularweight to the light chain of Ig). There is no evidence of otherprotein(s) in the purified preparation.

[0146] Identification of the mouse immunoglobulin isotype for the MG1antibody was carried out using the Pharmingen isotype typing kit, theresults of which are shown in Table 1. TABLE 1 ISOTYPE OF MG1IMMUNOGLOBULIN Anti- negative isotype Ab MG1 IgG MG1 IgG positivecontrol control Anti-IgG 1 0.019 0.025 0.768 0.023 Anti-IgG 2a 0.8710.808 0.435 0.000 Anti-IgG 2b 0.034 0.038 2.725 0.000 Anti-IgG 3 0.0290.035 0.208 0.000 Anti-IgM 0.027 0.076 1.073 0.000 Anti-IgA 0.026 0.0270.597 0.010 Anti-Ig Lκ 2.452 2.373 1.147 0.004 Anti-Ig Lλ 0.020 0.0190.807 0.000

[0147] The isotype of the purified MG1 immunoglobulin was determined byELISA. Plates pre-coated with anti-isotype antibody were blocked with 3%BSA in PBS, before the addition of the MG1 or control immunoglobulins.Alkaline phosphatase tagged rat anti-mouse Ig was used as the secondaryantibody. Reactions were read spectrophotometrically at 405 nm.Background levels of absorbance were determined for each row using wellstreated with only the immobilized anti-isotype antibody, the enzymetagged secondary antibody, and enzyme substrate. The positive controlused was mouse immunoglobulin cocktail. The negative control used wasmouse myeloma (Sp2/014) supernatant. The data shows that the MG1 isotypeis murine IgG2a.

[0148] The Reactivity of Clone 3.2.B11.b3 (MG1) with Established CellLines Derived from Hematopoietic Malignancies, HT-29 Cells, and NormalHuman Bone Marrow

[0149] Given the fact that the NSC1.1 cell line was derived from a humanhematopoietic stem cell population (CD34⁺), it was likely that the MG1antibody would recognize stem cells and/or immature (progenitor) cellsof the hematopoietic system. To test this, flow cytometry was performedon tumor cell lines isolated from malignancies of early stem cells,namely KG1a (an acute myelogenous leukemia), K562 (a chronic myelogenousleukemia), and HEL 92.1.7 (an erythroleukemia), and on normal human bonemarrow. From the flow cytometric analysis data (FIG. 3), it is evidentthat the MG1 monoclonal antibody, while recognizing 96.9% of NSC1.1cells, does not recognize any of the cells representing these threeleukemias. It is interesting to note that KG1a was the cell line used todiscover the CD34 antigen and since MG1 does not recognize KG1a, it ishighly probable that MG1 does not recognize the CD34 antigen. Further,the MG1 monoclonal antibody did not label HT-29 cells. Thisnon-hematopoietic cell line is derived from a human colonadenocarcinoma. These results suggest that MG1 is not recognizingantigens present on non-hematopoietic tissues.

[0150] Using fluorescein-labeled MG1 monoclonal antibody, whole humanbone marrow was analyzed by flow cytometry. The data from FIG. 4 givesfurther evidence that MG1 recognizes only a very small population (lessthan 1%) of cells within bone marrow. Furthermore, live-gating on CD34⁺cells indicated that less than 3% of CD34⁺ cells co-express MG1 antigen,suggesting a very small overlap of the two populations.

[0151] Utilizing immunomagnetic bead cell separation technology andpurified MG1 antibody, MG1⁺ bone marrow cells were recovered. When thecolony-forming potential of the MG1⁺-selected and the MG1-depletedpopulations were compared in CFU-GEMM assays, it was observed that theMG1⁺ cells were nearly 300-fold poorer at forming colonies than theMG1-depleted population (0.23 colonies per 10⁵ cells vs. 70±17 coloniesper 10⁵ cells, respectively. The lack of CFU activity exhibited by theMG1 cells with recombinant human IL-3, Il-6, and Epo in CFU-GEMM assaysgave further evidence to the primitive nature of the MG1⁺ cellpopulation. It is well established that quiescent hematopoietic stemcells, while retaining high proliferating potential, fail to respond toexogenous cytokines in CFU assays. (Berardi et al., Science 267:104-108(1995)). These cells however, will respond and proliferate in ex vivolong term culture initiating cell (LTCIC) assays. Berardi et al.,Science 267:104-108 (1995); Id.; Young et al., Blood 87:545-556 (1996).

[0152] Analysis of Specificity of the MG1 Monoclonal Antibody by WesternBlot

[0153] Western blot analysis was performed with two purposes in mind:First, to understand the scope of MG1's reactivity on different cellpopulations and second, to identify the ligand to which MG1 binds. Usinglysates prepared from the leukemia cell lines (KG1a, K562, and HEL92.1.7), western blot analysis was carried out, the results of which areshown in FIG. 5 (western blot using MG1 hybridoma supernatant-1/10dilution), FIG. 6 (western blot using MG1 hybridoma supernatant-1/100dilution) and FIG. 7 (western blot using purified MG1 IgG2a). The datafrom FIGS. 5-7 demonstrate that the only reactivity obtained was MG1binding to the NSC 1.1 lysate (lanes 2 and 9 of FIG. 5, lanes 1 and 9 ofFIG. 6, and lanes 2 and 9 of FIG. 7). These results confirm the earlierflow data (FIG. 3) in which the MG1 monoclonal antibody only bound tothe surface of the NSC1.1 cell and not to any of the cell linesmentioned above. The second observation to be made from FIGS. 5-7 isthat the protein that is recognized by MG1 is a high molecular weightprotein with a calculated (regression analysis) molecular weight ofapproximately 186 kDa±5%. Finally, a comparison of FIGS. 6 and 7 showthat the hybridoma supernatant and the purified IgG from the monoclonalhybridoma recognize the same protein in Western analyses. This confirmsthe derivation of the monoclonal.

[0154] Similar western blot experiments were carried out using celllysates from subpopulations of peripheral blood leukocytes enriched onself generated Percoll gradients. The blots were again probed withpurified MG1 monoclonal antibody (14 μg). The data from FIG. 8conclusively show that the MG1 antibody probe only recognizes NSC1.1lysate (lanes 1 and 10) and does not recognize proteins associated withcells isolated from normal peripheral blood. Again, the proteinrecognized by MG1 antibody appears to have a molecular weight ofapproximately 186 kDa±5%.

[0155] Therefore, combining all the data from the western blot analysesand the flow cytometry together, it is believed that the MG1 monoclonalantibody recognizes a cell surface antigen that is only expressed onrare cells within the hematopoietic environment. Furthermore, given theCFU data, it is believed that these cells reside within the primitivestem cell compartment of that tissue.

EXAMPLE 2 Characterization of the MG1 Antigen

[0156] The MG1 antigen is expressed on a small proportion of human bonemarrow cells, and this population overlaps with the CD34⁺ population.MG1 and CD34 doubly labeled cells represented only minor subpopulationsof the singly labeled cells. The results of flow analyses on bone marrowindicate that this overlapping population represents a small percentageof the CD34⁺ population, and also a small percentage of the MG1⁺population. In other words, few of the CD34 population also labels withMG1, and few of the MG1 population also labels with CD34. The fact thatindependent flow studies showed that MG1 did not recognize any of thesub populations of the peripheral blood leukocytes enriched on selfgenerated Percoll gradients, suggests that MG1 is not recognizing maturecells. Furthermore, when MG1-selected cells are placed into CFU-GEMMassays (with cytokines IL-3, IL-6, SCF and Epo), few if any colonies aredetected, whereas the MG1-depleted population does generate colonies.This indicates that MG1 is not recognizing committed progenitor cellseither. Recent results showing that high proliferative potential ofhematopoietic cells is associated with quiescence in CFU assays (Berardiet al., Science 267:104-108 (1995); Young et al., Blood 87:545-556(1996)) would suggest that the MG1+ population falls into the categoryof very primitive, quiescent cells.

[0157] Glycosylation Determination of the MG1 Antigen

[0158] In order to characterize the MG1 antigen more fully, the proteinwas subjected to deglycosylation using glyconases specific for N-linkedand O-linked carbohydrate moieties. It is evident from the data shown inFIG. 9 that the MG1 antigen is glycosylated and further, that thisglycosylation is predominantly comprised of N-linked sugars (lane 2).Treatment of the MG1 antigen with O-glyconase did not measurably (asdetectable by the resolving capability of this gel) decrease themolecular weight of the ligand (lane 3) indicating the lack of O-linkedsugars. This was further confirmed by treatment of the MG1 antigen witha mixture of the two enzymes, the results of which are shown in lane 1of FIG. 9, wherein the decrease in the molecular weight observed isequal to that of the N-glyconase treatment alone.

[0159] Purification of the MG1 Antigen

[0160] The MG1 antigen was partially purified by affinity columnchromatography. Lysates prepared from NSC1.1 cells were loaded onto animmunoaffinity column containing bound purified MG1 monoclonal antibody.The column was washed, and the bound protein was eluted with 0.1 Mglycine (pH 2.7) into 1.0 M Tris (pH 9.0). This eluate was concentratedby ultrafiltration, and examined by SDS PAGE. The size of the MG1antigen, as calculated by a regression analysis of the two proteinmarker lanes in FIG. 10 (non-prestained), was about 186 kDa±5%.

[0161] Reactivity with Laminin

[0162] The amino acid composition of the purified ligand was determined(see Table 2) and compared to other previously described proteins inavailable databases using two different computer programs (ExPASy andPropsearch). TABLE 2 AMINO ACID COMPOSITION FOR MG1 ANTIGEN Amino AcidPercent of Total Asx 12.81 Glx 15.47 Ser 5.34 His 3.37 Gly 11.64 Thr4.34 Ala 6.92 Pro 5.43 Tyr 2.56 Arg 4.12 Val 6.06 Met 0.71 Ile 4.92 Leu6.85 Phe 3.17 Lys 6.29

[0163] Results indicated that the MG1 antigen appeared to be a uniqueand novel protein. However, both programs found a low degree ofsimilarity with β1 subunit of human laminin. In order to examine thepossibility that the MG1 antigen could be the β1 subunit of humanlaminin, a Western blot analysis was conducted using NSC1.1 lysate and arabbit polyclonal antibody against human laminin (FIG. 11). The resultsindicated that the anti-laminin antibody did not recognize any proteinsin the NSC1.1 lysate suggesting that the MG1 antigen is not the β1subunit of human laminin.

[0164] N-terminal amino acid sequencing of purified MG1 antigen was alsoperformed. The partial amino acid sequence of the MG1 antigen is shownbelow (in standard three letter amino acid code):

[0165] ArgArgArgAlaLysGlnAsnGlnXaaGlyGluIle (SEQ ID NO. 1)

[0166] (where Xaa represents an undetermined residue). Since no otherpreviously described proteins showed significant sequence identity withthe amino acid composition and sequence of MG1 antigen, it was concludedthat the MG1 antigen represents a novel protein.

[0167] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

[0168] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 1 1 12 PRT Homo sapiens UNSURE 9 Unknown 1 Arg Arg Arg Ala Lys Gln AsnGln Xaa Gly Glu Ile 1 5 10

We claim:
 1. A method for selecting a population of human cellssubstantially enriched for hematopoietic cells expressing the MG1antigen, said method comprising the steps (a) contacting a human cellsuspension with an antibody that binds the MG1 antigen; and (b)recovering those cells which are bound by said antibody.
 2. The methodaccording to claim 1, wherein said human cell population is contactedwith at least a second antibody wherein said second antibody binds to anantigen other than said MG1 antigen.
 3. The method according to claim 1,wherein said human cell population is prepared from tissue selected fromthe group consisting of marrow and blood.
 4. The method according toclaim 1, wherein said antibody that binds said MG1 antigen is attachedto a solid phase matrix.
 5. The method according to claim 1, whereinsaid antibody that binds the MG1 antigen is the monoclonal antibodydesignated as MG1 and produced by the hybridoma cell line depositedunder ATCC Accession No. HB12232.
 6. The method according to claim 1,wherein said antibody that binds said MG1 antigen is labeled with adetectable label.
 7. A population of human cells substantially enrichedfor hematopoietic cells expressing the MG1 antigen produced according tothe method of claim
 1. 8. A method for detecting a malignant tumor cellin a sample of human cells, said method comprising contacting saidsample with an antibody that binds to an MG1 antigen and detecting thosecells which have bound said antibody, wherein binding of said antibodyto a cell is indicative of a malignant tumor cell.
 9. The methodaccording to claim 8, wherein said malignant cell is a cell associatedwith leukemia.